The Effect Of Photodynamic Therapy In Human Hepatocel Lular Carcinoma Cells SMMC-7721.

Objective:(1): To research the effect of photodynamic therapy with Hematoporphyrin Derivative in human hepatocellular carcinoma cells SMMC-7721..(2): To investigate the effect of survivin and caspase-3 in the apoptosis of hepatoma cells SMMC-7721 induced by Photodynamic Therapy(3): To evaluate the expression of vascular endothelial growth factor VEGF in photodynamic therapy in human hepatocellular carcinoma cells SMMC-7721..Methods:1, experimental groups: divided into four groups, an experimental group: HPD + PDT groups (photosensitizer + illumination); three control groups: control group (no X-ray, without photosensitizer) + HPD control group ( simply join the photosensitizer, the non-PT) + PDT group (simple X-ray, without photosensitizer).2,1.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide(MTT)assay was used to determine the cell survival rate of human hepatocellular carcinoma cells SMMC-7721.after photodynamic therapy with different concentrations of HPD (0.1,0.5,1.0,5.0,10 mg /L) incubated for 4 h, and a wavelength of 630 nm semiconductor laser irradiation( 5,10,15 J / cm~2), and then incubated for 24 h.3, Observed the human hepatocellular carcinoma cells SMMC-7721 after PDT at different time points after treatment of cell morphology under inverted phase contrast microscope ; 4, The apoptosis of human hepatocellular carcinoma cells SMMC-7721.after PDT was detected by Annexin V-FITC / PI double staining5, The expression of Survivin,Caspase-3,VEGF in human hepatocellular carcinoma cells SMMC-7721 after photodynamic therapy was detected by immunohistochemical staining .6, The expression levels of Survivin mRNA,Caspase-3 mRNA,VEGF mRNA was detected by RT-PCRResults:1, At the same conditions of laser, PDT treatment on cultured human hepatocellular carcinoma cells SMMC-7721 role (1) With the concentration of photosensitizer increases. But when the concentration of 5mg/L, the increase in drug concentration, the apoptosis rate increased is limited. (2) An the same conditions of photosensitizer, PDT treatment on cultured human hepatocellular carcinoma cells SMMC-7721's role: As the light dose increased. But when the concentration of 10J/cm~2, the further increase in light intensity, the apoptosis rate did not significantly increase. so,we screened 5mg/L, 10J / cm~2as the best experimental parameters,2, The appearance of morphological changes of human hepatocellular carcinoma cells SMMC-7721 at different times after PDT effect after 6 h part of the PANC-1 cells, cell shrinkage began to appear, 12 h later to narrow the vast majority of cells into round, some small Cell membrane blistering; to 24 h, a large number of cells can be seen floating in the culture medium, the only remaining form of a very small number of normal cells.3,The apoptosis rate of human hepatocellular carcinoma cells SMMC-7721 after PDT according to the best experimental parameters, the apoptosis of human hepatocellular carcinoma cells SMMC-7721 were detected by flow cytometry (FCM): HPD + PDT experimental group reached(33.42±5.52) %, significantly higher than that in HPD( 9.20±2.50) %, or PDT( 7.40±0.75 ) % and the blank ( 6.67±1.25) % (P <0.05), with control groups, no significant difference (P> 0.05)4, The expression of VEGF protein levels after PDT The human hepatocellular carcinoma cells SMMC-7721 immunohistochemical staining :optical microscope observation PDT group, HPD group and blank control group shows that many cell cytoplasm brownish yellow, high-powered microscope, the cytoplasm there are large brown region, while the HPD + PDT pale-brown staining within the experimental group significantly reduced cytoplasm; Survivin protein and VEGF protein in each group the results of immunohistochemical expression: HPD + PDT experimental group, the mean optical density of VEGF protein than the control group significantly lower than (P <0.05), while Caspase-3 contraryly ,the PDT group, HPD group and blank control group, three groups no statistically significant difference (P> 0.05) .5, The expression levels of VEGF mRNA after PDT Uunder the best experimental parameters ,RT-PCR showed the expression levels of Survivin mRNA and VEGF mRNA after PDT of HPD + PDT experimental group was significantly lower than the control group (P <0.05) , while Caspase-3 contraryly,and the three control group, no significant difference (P> 0.05).Conclusion:1, Photodynamic therapy can induce apoptosis and necrosis of human hepatocellular carcinoma cells SMMC-7721 and this effect was relatived to the HPD concentration and the light output power .2, The apoptosis induced by photodynamic therapy in human hepatoma cells SMMC7721, may be related to the expression inhibition of Survivin protein , and directly or indirectly promote the expression of Caspase-3. 3, The expression of VEGF in human hepatocellular carcinoma cells SMMC-7721 can be inhibited by photodynamic therapy...